Ionic dependence of sodium currents in squid axons analyzed in terms of specific ion “channel” interactions (original) (raw)
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The Journal of General Physiology, 1969
Isolated giant axons were voltage-clamped in seawater solutions having constant sodium concentrations of 230 mM and variable potassium concentrations of from zero to 210 mM. The inactivation of the initial transient membrane current normally carried by Na+ was studied by measuring the Hodgkin-Huxley h parameter as a function of time. It was found that h reaches a steady-state value within 30 msec in all solutions. The values of h∞, τh, αh,and ßh as functions of membrane potential were determined for various [Ko]. The steady-state values of the h parameter were found to be inversely related, while the time constant, τh, was directly related to external K+ concentration. While the absolute magnitude as well as the slopes of the h∞ vs. membrane potential curves were altered by varying external K+, only the magnitude and not the shape of the corresponding τh curves was altered. Values of the two rate constants, αh and ßh, were calculated from h∞ and τh values. αh is inversely related to...
Unidirectional sodium and potassium fluxes through the sodium channel of squid giant axons
Biophysical Journal, 1982
Unidirectional 22Na-traced sodium influx or 42K-traced potassium efflux across the membranes of voltage-clamped squid giant axons was measured at various membrane potentials under bi-ionic conditions. Tetrodotoxin almost entirely eliminated the extra K+ efflux induced by short repetitive depolarizations in the presence of tetraethylammonium or 3,4-diaminopyridine. A method of determining the voltage dependence of the unidirectional flux through voltage-gated channels is described. This technique was used to obtain the unidirectional flux-voltage relation for the sodium channel in bi-ionic and single-ion conditions. It allows the determination of the unidirectional flux at the zero-current potential which, for influx, was found to be approximately 20% of the value measured 80 mV negative to the zero-current potential. The unidirectional flux ratio under bi-ionic conditions was also measured and the flux ratio exponent found to average 1.15 with an external sodium and an internal potassium solution. A three-barrier, two-site, multi-occupancy model previously obtained for other conditions was found to predict a similar non-unity average for the flux ratio exponent. It is also shown that some single-occupancy models can predict non-unity values for the flux ratio exponent in bi-ionic conditions.
The Journal of General Physiology, 1985
Inactivation of Na channels has been studied in voltage-clamped, internally perfused squid giant axons during changes in the ionic composition of the intracellular solution. Peak Na currents are reduced when tetramethylammonium ions (TMA+) are substituted for Cs ions internally. The reduction reflects a rapid, voltage-dependent block of a site in the channel by TMA+. The estimated fractional electrical distance for the site is 10% of the channel length from the internal surface. Na tail currents are slowed by TMA+ and exhibit kinetics similar to those seen during certain drug treatments. Steady state INa is simultaneously increased by TMA+, resulting in a "cross-over" of current traces with those in Cs+ and in greatly diminished inactivation at positive membrane potentials. Despite the effect on steady state inactivation, the time constants for entry into and exit from the inactivated state are not significantly different in TMA+ and Cs+. Increasing intracellular Na also r...
The Journal of General Physiology, 1969
Giant axons were voltage-clamped in solutions of constant sodium concentration (230 mM) and variable potassium concentrations (from 0 to 210 mM). The values of the peak initial transient current, I(p), were measured as a function of conditioning prepulse duration over the range from less than 1 msec to over 3 min. Prepulse amplitudes were varied from E(m) = -20 mv to E(m) = -160 mv. The attenuation of the I(p) values in high [K(o)] was found to vary as a function of time when long duration conditioning potentials were applied. In both high and low [K(o)], I(p) values which had reached a quasi-steady-state level within a few milliseconds following a few milliseconds of hyperpolarization were found to increase following longer hyperpolarization. A second plateau was reached with a time constant of about 100-500 msec and a third with a time constant in the range of 30 to 200 sec. The intermediate quasi-steady-state level was absent in K-free ASW solutions. Sodium inactivation curves, normalized to I(pmax) values obtained at either the first or second plateaus, were significantly different in different [K(o)]. The inactivation curves, however, tended to superpose after about 1 min of hyperpolarizing conditioning. The time courses and magnitudes of the intermediate and very slow sodium conductance restorations induced by long hyperpolarizing pulses are in agreement with those predicted from the calculated rates and magnitudes of [K(+)] depletion in the space between the axolemma and the Schwann layer.
The early phase of sodium channel gating current in the squid giant axon
European Biophysics Journal, 1992
A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligoforbesii. This component is characterized by relaxation time constants typically shorter than 25 #s. The charge displaced accounts for about 10% (or 2 nC/cm 2) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above +20 mV and below -100 inV. This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of I e-and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.
The conductance and density of sodium channels in the cut-open squid giant axon
The Journal of physiology, 1986
Non-stationary Na current fluctuations in small voltage-clamped patches of cut-open squid giant axon were analysed by an ensemble-average technique to yield the single Na channel conductance gamma Na and the Na channel density in the patch. gamma Na appeared to be voltage independent over the range -30 to +40 mV and had a mean value of 4.4 +/- 1.1 pS in 514 mM-Na/20 mM-Na at 5 kHz band width and temperature between 3.5 and 5.0 degrees C. gamma Na did not change significantly at band widths to 20 kHz. gamma Na in reduced Na solutions, 103 mM-Na/4 mM-Na, at 3.5-5.0 degrees C had a mean value of 1.2 +/- 0.3 pS. Internal solutions containing 50 mM-tetraethylammonium (TEA) depressed both gamma Na and the mean Na currents by roughly the same factor, compared with solutions without TEA. The reduced gamma Na had a mean value of 2.2 +/- 0.7 pS. The mean Na channel density in the standard 514 mM-Na/20 mM-Na solution was estimated to be 180 +/- 100 microM-2. The densities in the other solution...
Sodium flux ratio in voltage-clamped squid giant axons
The Journal of General Physiology, 1981
The sodium flux ratio across the axolemma of internally perfused, voltage-clamped giant axons of Loligo pealei has been measured at various membrane potentials. The flux ratio exponent obtained from these measurements was about unity and independent of membrane voltage over the 50 mV range from about -20 to ł mV. These results, combined with previous measurements of ion permeation through sodium channels, show that the sodium channel behaves like a multi-ion pore with two ion binding sites that are rarely simultaneously occupied by sodium.
The Journal of Physiology, 1981
The efflux of Na in dialysed axons of the squid has been used to monitor the sidedness of the interactions of the Na pump with Na+ ions, K+ ions and ATP. The axons were under conditions such that most of the Na efflux went through the Na pump by means of a complete cycle of ATP hydrolysis. 2. With 310 mM-Kj, 70 mM-Nat and 10 mM-K+ artificial sea water (ASW) more than 97 % of the Na efflux was abolished by removal of ATP. The efflux of Na was stimulated by ATP with a Kj of about 200 /1M. This is similar to the KI of 150 /SM found for the ATP dependence of a ouabain-sensitive Na,K-ATPase activity in membrane fragments isolated from squid optical nerves. 3. A 100-fold reduction in the ATP concentration (from 3-5 mm to 30-50 /M) increased the apparent affinity of the Na pump for K+ about 8-fold. In addition, the maximal rate of K+-stimulated Na efflux was reduced by a similar factor. Analogous results were seen in axons dialysed with 310 mM-Kt or without Kt. 4. The relative effectiveness of external monovalent cations as activators of the Na efflux was a function of the ATP concentration inside the axon. With 3-5 mM-ATP the order of effectiveness was K+ > NH+ > Rb+. With 30-50 ,uM-ATP the sequence was NH4+ > K+ > Rb+. These results were not affected by the removal of Kt. 5. When the ATP concentration was 3 mm and the Nat concentration 70 mm, the removal of Kt produced a slight and reversible increase in the total efflux ofNa (15 %) and no change in the ATP-dependent Na efflux. When the ATP concentration was reduced to 30-50 /,tM, or the Nat concentration lowered to 5-10 mm, the removal of Kt reversibly increased the total and the ATP-dependent efflux of Na. The largest increase in Na efflux was seen when both ATP and Nat were simultaneously reduced. The ATP-dependent extra Na efflux resulting from the exclusion of Kt was abolished by 10-4 M-ouabain in the sea waters. 6. The increase in the ATP-dependent Na efflux observed in axons dialysed with 0 Kt+ 10 mM-K+ ASW was not seen in axons perfused with 310 mM-Kt+450 mr-K+ ASW. However, both experimental conditions gave rise to a similar (and small) ATP-independent and ouabain-insensitive efflux of Na. This indicates that the effects